Neonatal cytokines associated with infant overweight and obesity at 1 year of age.

OBJECTIVE: The incidence of childhood overweight and obesity has been increasing in recent years. Immune dysregulation has been demonstrated as a condition related to childhood obesity. Whether the neonatal immune status is related to infant overweight and obesity at 1 year of age is unclear. METHODS: To explore the relationship between neonatal cytokines and infant overweight and obesity, we conducted a prospective study in Suzhou Municipal Hospital Affiliated to Nanjing Medical University from 2015 to 2016. 514 neonates were recruited and their dried blood spots were collected after birth. Infants were grouped into normal size groups and overweight and obesity groups based on BMI at 1 year of age. 27 neonatal cytokines levels were compared between the two groups. RESULTS: 370 infants were included in final analysis. Granulocyte colony stimulating factor (GCSF), interleukin-17A (IL17A) and platelet derived growth factor-BB (PDGF-BB) levels were independently associated with childhood overweight and obesity (OR =1.27, 95%CI 1.03, 1.57; OR =1.29, 95%CI: 1.06, 1.60; OR =0.69, 95%CI: 0.49, 0.96). Additionally, neonatal GCSF and IL17A levels were positively associated with increased BMI (ß = 0.11, 95%CI: 0.02, 0.19; ß = 0.07, 95%CI 0.01, 013) and BMI z-scores (ß = 0.10, 95%CI: 0.02, 0.18; ß = 0.06, 95%CI 0.01, 0.13). Neonatal PDGF-BB levels were negatively associated with BMI (ß = -0.12, 95%CI: -0.23, -0.01) and BMI z-scores (ß = -0.12, 95%CI: -0.23, -0.01). The inverse probability weighting (IPW) was performed to account for potential selection bias of this study, and the results were consistent with the above mentioned findings. CONCLUSIONS: Neonatal GCSF, IL17A and PDGF-BB levels were correlated with infant overweight and obesity at 1 year of age, suggesting that early life immune status play a significant role of late obesity.

Investigating the Metabolic Model in Preterm Neonates by Tandem Mass Spectrometry: A Cohort Study.

The changes of metabolite profiles in preterm birth have been demonstrated using newborn screening data. However, little is known about the holistic metabolic model in preterm neonates. The aim was to investigate the holistic metabolic model in preterm neonates. All metabolite values were obtained from a cohort data of routine newborn screening. A total of 261 758 newborns were recruited and randomly divided into a training subset and a testing subset. Using the training subset, 949 variates were considered to establish a logistic regression model for identifying preterm birth (<37 weeks) from term birth (≥37 weeks). Sventy-two variates (age at collection, TSH, 17α-OHP, proline, tyrosine, C16:1-OH, C18:2, and 65 ratios) entered into the final metabolic model for identifying preterm birth from term birth. Among the variates entering into the final model of PTB [Leucine+Isoleucine+Proline-OH)/Valine (OR=38.36], (C3DC+C4-OH)/C12 (OR=15.58), Valine/C5 (OR=6.32), [Leucine+isoleucine+Proline-OH)/Ornithine (OR=2.509)], and Proline/C18:1 (OR=2.465) have the top five OR values, and [Leucine+Isoleucine+Proline-OH)/C5 (OR=0.05)], [Leucine+Isoleucine+Proline-OH)/Phenylalanine (OR=0.214)], proline/valine (OR=0.230), C16/C18 (OR=0.259), and Alanine/free carnitine (OR=0.279) have the five lowest OR values. The final metabolic model had a capacity of identifying preterm infants with >80% accuracy in both the training and testing subsets. When identifying neonates ≤32 weeks from those >32 weeks, it had a robust performance with nearly 95% accuracy in both subsets. In summary, we have established an excellent metabolic model in preterm neonates. These findings could provide new insights for more efficient nutrient supplements and etiology of preterm birth.

3-Methylcrotonyl-CoA carboxylase deficiency newborn screening in a population of 536,008: is routine screening necessary?

Objective To evaluate whether 3-methylcrotonyl-CoA carboxylase deficiency (3-MCCD) should be routinely screened in newborns. Methods Dried blood spots (DBS) were collected and analyzed by tandem mass spectrometry (TMS). Blood samples were collected from infants with positive 3-MCCD results. Targeted sequencing was performed using the extended panel for inherited metabolic diseases to detect 306 genes. The sequencing libraries were quantified and used for massively parallel sequencing on the Illumina HiSeq 2500 platform. Results A total of 536,008 infants underwent newborn screening (NBS) and 14 cases of 3-MCCD were diagnosed. The incidence of 3-MCCD in Jiangsu province was 1:38,286. During the last 3 years of follow-up, none of the subjects with 3-MCCD exhibited obvious clinical symptoms. Only two children had mild feeding difficulties and vomiting. Eleven patients had complex variants of the MCCC1 gene, and three patients had mutations in MCCC2. In total, 17 types of MCCC1 or MCCC2 variants were found, and c.639 + 2t > a was the most common mutation. Conclusions As far as the current results are concerned, 3-MCCD may be benign in Jiangsu province. However, additional investigations and a longer follow-up period are necessary to decide whether NBS of 3-MCCD is necessary or not.

Expanded Newborn Screening for Inborn Errors of Metabolism by Tandem Mass Spectrometry in Suzhou, China: Disease Spectrum, Prevalence, Genetic Characteristics in a Chinese Population.

Expanded newborn screening for inborn errors of metabolism (IEMs) by tandem mass spectrometry (MS/MS) could simultaneously analyze more than 40 metabolites and identify about 50 kinds of IEMs. Next generation sequencing (NGS) targeting hundreds of IMEs-associated genes as a follow-up test in expanded newborn screening has been used for genetic analysis of patients. The spectrum, prevalence, and genetic characteristic of IEMs vary dramatically in different populations. To determine the spectrum, prevalence, and gene mutations of IEMs in newborns in Suzhou, China, 401,660 newborns were screened by MS/MS and 138 patients were referred to genetic analysis by NGS. The spectrum of 22 IEMs were observed in Suzhou population of newborns, and the overall incidence (excluding short chain acyl-CoA dehydrogenase deficiency (SCADD) and 3-Methylcrotonyl-CoA carboxylase deficiency (3-MCCD)) was 1/3,163. The prevalence of each IEM ranged from 1/401,660 to 1/19,128, while phenylketonuria (PKU) (1/19,128) and Mild hyperphenylalaninemia (M-HPA) (1/19,128) were the most common IEMs, followed by primary carnitine uptake defect (PCUD) (1/26,777), SCADD (1/28,690), hypermethioninemia (H-MET) (1/30,893), 3-MCCD (1/33,412) and methylmalonic acidemia (MMA) (1/40,166). Moreover, 89 reported mutations and 51 novel mutations in 25 IMEs-associated genes were detected in 138 patients with one of 22 IEMs. Some hotspot mutations were observed for ten IEMs, including PAH gene c.728G > A, c.611A > G, and c.721C > T for Phenylketonuria, PAH gene c.158G > A, c.1238G > C, c.728G > A, and c.1315+6T > A for M-HPA, SLC22A5 gene c.1400C > G, c.51C > G, and c.760C > T for PCUD, ACADS gene c.1031A > G, c.164C > T, and c.1130C > T for SCAD deficiency, MAT1A gene c.791G > A for H-MET, MCCC1 gene c.639+2T > A and c.863A > G for 3-MCCD, MMUT gene c.1663G > A for MMA, SLC25A13 gene c.IVS16ins3Kb and c.852_855delTATG for cittrullinemia II, PTS gene c.259C > T and c.166G > A for Tetrahydrobiopterin deficiency, and ACAD8 gene c.1000C > T and c.286C > A for Isobutyryl coa dehydrogenase deficiency. All these hotspot mutations were reported to be pathogenic or likely pathogenic, except a novel mutation of ACAD8 gene c.286C > A. These mutational hotspots could be potential candidates for gene screening and these novel mutations expanded the mutational spectrum of IEMs. Therefore, our findings could be of value for genetic counseling and genetic diagnosis of IEMs.

New Ratios for Performance Improvement for Identifying Acyl-CoA Dehydrogenase Deficiencies in Expanded Newborn Screening: A Retrospective Study.

Some success in identifying acyl-CoA dehydrogenase (ACAD) deficiencies before they are symptomatic has been achieved through tandem mass spectrometry. However, there has been several challenges that need to be confronted, including excess false positives, the occasional false negatives and indicators selection. To select ideal indicators and evaluate their performance for identifying ACAD deficiencies, data from 352,119 newborn babies, containing 20 cases, were used in this retrospective study. A total of three new ratios, C4/C5DC+C6-OH, C8/C14:1, and C14:1/C16-OH, were selected from 43 metabolites. Around 903 ratios derived from pairwise combinations of all metabolites via multivariate logistic regression analysis were used. In the current study, the regression analysis was performed to identify short chain acyl-CoA dehydrogenase (SCAD) deficiency, medium chain acyl-CoA dehydrogenase (MCAD) deficiency, and very long chain acyl-CoA dehydrogenase (VLCAD) deficiency. In both model-building and testing data, the C4/C5DC+C6-OH, C8/C14:1 and C14:1/C16-OH were found to be better indicators for SCAD, MCAD and VLCAD deficiencies, respectively, compared to [C4, (C4, C4/C2)], [C8, (C6, C8, C8/C2, C4DC+C5-OH/C8:1)], and [C14:1, (C14:1, C14:1/C16, C14:1/C2)], respectively. In addition, 22 mutations, including 5 novel mutations and 17 reported mutations, in ACADS, ACADM, and ACADL genes were detected in 20 infants with ACAD deficiency by using high-thorough sequencing based on target capture. The pathogenic mutations of c.1031A > G in ACADS, c.449_452delCTGA in ACADM and c.1349G > A in ACADL were found to be hot spots in Suzhou patients with SCAD, MCAD, and VLCAD, respectively. In conclusion, we had identified three new ratios that could improve the performance for ACAD deficiencies compared to the used indicators. We considered to utilize C4/C5DC+C6-OH, C8/C14:1, and C14:1/C16-OH as primary indicators for SCAD, MCAD, and VLCAD deficiency, respectively, in further expanded newborn screening practice. In addition, the spectrum of mutations in Suzhou population enriches genetic data of Chinese patients with one of ACAD deficiencies.

Application of Next-Generation Sequencing Following Tandem Mass Spectrometry to Expand Newborn Screening for Inborn Errors of Metabolism: A Multicenter Study.

This study explored the effectiveness of expanding newborn screening (NBS) by tandem mass spectrometry (TMS) and gene diagnosis by next-generation sequencing (NGS). First, we described the characteristics of gene variants in Jiangsu Province. We collected clinical data from three NBS centers. All infants followed a unified screening and diagnosis process. After obtaining informed consent, dried blood spots (DBSs) were collected and analyzed by TMS. If the results fell outside of the cut-off value, repeat analysis was performed. If the re-test results remained abnormal, the infant was recalled for further assessment. We performed targeted sequencing using the extended edition panel of inborn errors of metabolism (IEM) to detect 306 genes using the Illumina HiSeq 2500 platform. A total of 536,008 babies underwent NBS by TMS in three NBS centres. In total, 194 cases were eventually diagnosed with various types of inherited metabolic diseases, with an overall incidence of 1/2763. There were 23 types of diseases, including ten amino acid disorders (43.5%), eight organic acidaemias (34.8%) and five fatty acid oxidation defects (21.7%). In these infants, we clearly identified variants of disease-causing genes by next-generation sequencing (NGS). Most had two variants and others had one or three variants: 88% of gene variants were heterozygous and 12% were homozygous. There is a certain incidence of IEM in Jiangsu Province and it is necessary to carry out screening for 27 diseases. Meanwhile, NGS combined with TMS offers an enhanced plan for NBS for IEM.

Development and validation of a fetal genotyping assay with potential for noninvasive prenatal diagnosis of hereditary hearing loss.

OBJECTIVE: Inherited non-syndromic hearing loss (NSHL) is a common sensory disorder that afflicts otherwise healthy individuals. The aim of the study was to evaluate the performance of circulating single molecule amplification and re-sequencing technology (cSMART) for non-invasive prenatal testing (NIPT) of NSHL. METHOD: Neonatal inheritance of NSHL mutations was determined from bloodspots using SNaPshot genotyping. NIPT of cell-free DNA for fetal NSHL mutations in the GJB2, GJB3 and SLC26A4 genes was performed by a multiplex cSMART assay. The percentage of mutant alleles was used to deduce fetal DNA fractions and assign fetal genotypes. RESULTS: A total of 25 plasma samples selected with different fetal NSHL genotypes were coded and retrospectively analyzed by NIPT. Three normal fetuses, 18 carrier fetuses comprising seven GJB2 109G>A, four GBJ2 235delC, three GJB2 299-300delAT and four SLC26A4 IVS7-2A>G heterozygotes and four affected fetuses comprising two GJB2 109G>A homozygotes, one GBJ2 235delC homozygote and one compound GJB2 235delC/299-300delAT heterozygote were identified. All 25 fetal genotypes determined by the cSMART assay were concordant with neonatal genotypes. CONCLUSION: The cSMART assay applied to cell-free DNA isolated from maternal plasma of pregnant women is highly accurate for calling correct fetal NSHL genotypes. © 2016 John Wiley & Sons, Ltd.

SNPscan as a high-performance screening tool for mutation hotspots of hearing loss-associated genes.

In the present study, to assess the feasibility of the SNPscan technique for mutation screening in patients with nonsyndromic hearing loss (NSHL) and neonatus in China, the SNPscan technique was compared with the SNaPshot screening system. Chinese patients (162) with NSHL were used as the experimental group and 276 children without HL were used as the control group, respectively. SNPscan detected molecular defects in 112 patients (68.5%). In this technique, 83 patients (51.2%) with homozygous or compound heterozygous had confirmed molecular etiology in the GJB2, SLC26A4, and MT-RNR1 genes. By contrast, SNaPshot detected molecular defects in 103 patients (63.6%). In this method, 72 subjects (44.4%) with HL were confirmed to have NSHL caused by these mutations. This study demonstrates that SNPscan performs equally well or better than earlier routine genotyping method for genetic hearing loss, with possibility of detecting a larger variety of mutation.

Methylenetetrahydrofolate reductase polymorphisms at 3'-untranslated region are associated with susceptibility to preterm birth.

BACKGROUND: Etiology and mechanism of preterm birth (PTB) is complicated. Genetic susceptibility is one of the key factors involved in the pathogenic mechanism underlying PTB. METHODS: A subset of single nucleotide polymorphisms (SNPs) selected by bioinformatics approach from 3'-untranslated region (3'-UTR) of methylenetetrahydrofolate reductase (MTHFR) gene were subjected to SNaPshot analysis in a case-control study. Three SNPs (rs45451599, rs1537515, rs1537516) were simultaneously tested in one tube, among 1,135 DNA samples including 480 PTBs and 655 term controls. RESULTS: Two perfectly correlated (r(2)=1) SNPs, rs1537515 and rs1537516, were found significantly associated with PTB susceptibility [P=0.012; OR: 0.65; 95% confidence interval (CI), 0.47-0.91]. The frequencies of the minor alleles were lower in PTB cases than in controls, which the frequencies were 0.066 in PTB cases and 0.095 in controls. G and T allele frequencies of rs1537515 were the same with rs1537516 (P=0.011; OR: 0.666; 95% CI, 0.49-0.91). Rs45451599 was not found associated with PTB (P=0.52; OR: 0.76; 95% CI, 0.33-1.74). The 18-25 nucleotides in length of microRNAs (miRNAs) which can regulate gene expressions are involved in binding partial complementary sequences within 3'-UTR. The two loci are at 3'-UTR of MTHFR mRNA. Rs1537516 is a potential target of miR-1304-3p, while rs1537515 is miR-1224-3p and miR-3150-5p. CONCLUSIONS: In conclusion, rs1537515 and rs1537516 within the 3'-UTR of the MTHFR gene may be associated with susceptibility to PTB.

[Multicenter investigation on the impact of newborn infants' gestational age and birth weight on the level of 17α-hydroxyprogesterone].

OBJECTIVE: To investigate the correlation of gestational age and birth weight with 17α-hydroxyprogesterone (17α-OHP) levels, and with results of adrenal hyperplasia newborn screening. METHOD: Using time-resolved fluorescence immunoassay, the authors measured concentrations of heel blood 17α-OHP by newborn dried blood spots on filter paper which included 29 hospitals newborns of Wujiang, Taicang, Zhangjiagang, Kunshan, and Suzhou, where there were 118 050 infants in total who had accurate gestational age and birth weight (62 490 males, 55 560 females). According to the classification by gestational age, there were 4 693 premature infants, 113 300 term infants and 57 overdue infants. According to the classification by birth weight, there were 4 172 infants with weight < 2 500 g, and 113 878 infants weight ≥ 2 500 g. And, in all premature infants, gestational age of 189 infants was < 32 weeks, 2 277 infants less than 36 weeks but ≥ 32 weeks, and 2 227 infants less than 37 weeks but not less than 36 weeks. Neonatal heel blood concentration of 17α-OHP was measured by dissociation enhanced lanthanide fluorescence immunoassay (DELFIA), and the correlation between 17α-OHP and gestational age or birth weight was retrospectively analyzed by using Spearman test. RESULT: The distribution of 17α-OHP levels was skew. The 17α-OHP levels decreased significantly from very preterm births, moderately preterm, later period preterm to term infants [19.21 (8.07, 24.00), 12.35 (6.81, 18.00), 8.58 (5.66, 13.80), 5.60 (3.57, 8.51) , 3.34 (2.58, 5.23) nmol/L; 479.42, 62.25, 36.24, 23.30, 13.73 nmol/L;P all = 0.000]. The 17α-OHP levels decreases from very low birth weight (VLBW), extremely low birth weight (ELBW), low birth weight (LBW), normal birth weight to macrosomia [5.24 (3.24, 8.96) , 11.30 (6.84, 22.95) , 8.50 (5.28, 14.90) , 5.66 (3.61, 8.62) , 5.38 (3.40, 8.11) nmol/L; 485.26, 125.18, 39.50, 23.80, 22.15 nmol/L; P = 0.000 for all comparison]. Neonatal 17α-OHP levels and gestational age, body weight was significantly negatively correlated respectively -16.40 and -10.10 (P both = 0.000) by using Spearman test. Neonatal 17α-OHP levels and gestational age, body weight were binomially distributed, and the formulae were y = 0.105 5x²-2.457 6x + 17.689, R² = 0.980 3 and y = 0.411x²-3.988x+14.75, R² = 0.983. Little preterm infants, preterm infants and term infants in low birth weight infants 17α-OHP levels were significantly higher than non-low birth weight infants [11.20 (6.01, 18.90) vs 9.05 (5.85, 14.90) nmol/L, 9.76 (4.32, 10.35) vs 5.59 (3.56, 8.48) nmol/L, P all = 0.000]. CONCLUSION: Neonatal 17α-OHP levels and gestational age, body weight was significantly negatively correlated; in order to improve the accuracy and sensitivity, cut-off value of neonatal 17α-OHP should be adjusted according to gestational age and weight.

Confined placental origin of the circulating cell free fetal DNA revealed by a discordant non-invasive prenatal test result in a trisomy 18 pregnancy.

BACKGROUND: Non-invasive prenatal testing (NIPT) by massively parallel sequencing is a useful clinical test for the detection of common fetal aneuploidies. While the accuracy of aneuploidy detection can approach 100%, results discordant with the fetus are occasionally reported. In this study we investigated the basis of a discordant T21 positive and T18 negative NIPT result associated with a T18 fetus confirmed by karyotyping. METHODS: Massively parallel sequencing was used to detect fetal DNA in maternal circulating plasma. The parental origin and nature of the fetal and placental aneuploidies were investigated by quantitative fluorescent PCR of short tandem repeat (STR) sequences and by copy number variation (CNV) sequencing. RESULTS: There was no evidence of T21 maternal mosaicism, T21 microchimerism or a vanishing twin to explain the discordant NIPT result. However, examination of multiple placental biopsies showed both T21 and T18 mosaicism, including one confined region with a significantly higher proportion of T21 cells. Based on fetal DNA fractions and average mosaicism levels, the effective T21 and T18 fetal DNA fractions should have been sufficient for the detection of both trisomies. CONCLUSIONS: In this pregnancy, we speculate that confined placental region(s) with higher proportions of T21 cells were preferentially releasing fetal DNAs into the maternal circulation. This study highlights placental mosaicism as a significant risk factor for discordant NIPT results.

Association of SNPs in genes involved in folate metabolism with the risk of congenital heart disease.

OBJECTIVE: To investigate the association of 12 single nucleotide polymorphisms (SNPs) in folate metabolic genes with congenital heart disease (CHD). METHODS: A total of 160 children with CHD and 188 control children were enrolled. Twelve SNPs related to folate metabolism, including CBS-C699T, DHFR-c594 + 59del19, FOLH1-T1561C, CBS-C699T, DHFR-c594 + 59del19, GSTO1-C428T, MTHFD-G878A and -G1958A, MTHFR-C677T and -A1298C, MTR-A2756G, MTRR-A66G, NFE2L2-ins1 + C11108T, RFC1-G80A, TCN2-C776T and TYMS-1494del6, were genotyped by SNaPShot genotyping technology and confirmed by Sanger sequencing. RESULTS: There were two SNPs including NFE2L2-ins1 + C11108T and GST01-C428T and two compound mutants for (MTHFD-G1958A, MTHFR-C677T and MTR-A2756G) and (MTHFD-G1958A, RFC1-G80A and MTR-A2756G), which might increase the risk of CHD, and DHFR-c594 + 59del19 might decrease the risk of CHD. The CT genotype of NFE2L2-ins1 + C11108T, OR = 2.15 (95% CI = [1.07, 4.32], p < 0.05). The CT + TT genotype of NFE2L2-ins1 + C11108T, OR = 1.98 (95% CI = [1.00, 3.93], p < 0.05). The TT genotype of GST01-C428T, OR = 3.49, (95CI% = [1.06, 11.5], p < 0.05). The GG genotype of DHFR-c594 + 59del19, OR = 0.46 (CI% = [0.24, 0.87], p < 0.05). The AG + GG genotype of DHFR-c594 + 59del19, OR = 0.53 (CI% = [0.29, 0.96], p < 0.05). The ratios of the two compound mutants for (MTHFD-G1958A, MTHFR-C677T and MTR-A2756G) and (MTHFD-G1958A, RFC1-G80A and MTR-A2756G) in CHD are higher than that in control, p < 0.05 (OR = 2.968, 95% CI = [1.022, 8.613]). CONCLUSIONS: The CT genotype of NFE2L2-ins1 + C11108T and the TT genotype of GST01-C428T are susceptible factors for CHD. The AG, GG and (AG + GG) genotypes of DHFR-c594 + 59del19 are protective genotypes for CHD. Compound mutants for (MTHFD-G1958A, MTHFR-C677T and MTR-A2756G) and (MTHFD-G1958A, RFC1-G80A and MTR-A2756G) may increase the risk of CHD.

[Killing effect of sodium abietate on adult worms of Schistosoma japonicum in vitro].

OBJECTIVE: To observe the killing effect of sodium abietate on adult male and female worms of Schistosoma japonicum in vitro. METHODS: The mice infected with cercariae of S. japonicum were sacrificed and perfused five weeks later, the adult worms obtained by the portal perfusion method, were cultivated in DMEM medium containing different concentrations of sodium abietate for 3 days, except the controls, then the worms were observed for the death and motility reducing. The worms were stained by hydrochloric acid carmine for the detection of the changes, and the protein of the worms was detected by using the ultraviolet ray-absorption and Bradford method. RESULT: After the treatment of sodium abietate, the mortality and motility reducing rate of adult worms were higher significantly than the controls; the effect of sodium abietate on male worms was more obvious than on female worms. The male worms' intestinal canal enlarged and appeared black or brown bands or spots after the treatment. The contents of the intestine of female worms were distributed asymmetrically, and the shape of some worms' ovaries was anomalism and the coloring was asymmetrical. Compared with the control group, the protein of adult male and female worms were reduced (P < 0.05). CONCLUSION: Sodium abietate could kill adult worms of S. japonicum in vitro. It may affect the protein metabolism of the worms.